Eicosapentaenoic acids and methods for their production

ABSTRACT

This invention describes a process for producing single cell edible oils containing eicosapentaenoic acid (EPA) from heterotrophic diatoms. The diatoms are cultivated in a fermentor in a nutrient solution containing nitrogen and silicate. Withdrawal of the nitrogen followed by withdrawal of the silicate induces the diatoms to synthesize large quantities of edible oil containing EPA which subsequently is recovered. The edible oil, and uses for it, also form parts of this invention as do mutant diatoms capable of producing large quantities of EPA.

BACKGROUND OF THE INVENTION

This invention relates to edible oils containing omega-3-fatty acids, inparticular eicosapentaenoic acid (EPA). The invention also relates tomethods of producing EPA in commercially viable yields.

Omega-3-fatty acids are known to be beneficial in reducing the incidenceof coronary heart disease. The metabolism of omega-3-fatty acids is notunderstood. Thus, although these acids are known to have beneficialeffects, precise clinical dosages and efficacy are not known.

Omega-3-fatty acids, including EPA, have been found in the oils of coldwater marine fish. Indeed, this is the primary source of commerciallyavailable EPA. It is believed that the omega-3-fatty acids found in fishoriginate from phytoplankton which are at the base of the marine foodchain. The belief is due to the fact that many phytoplankton species arefound to contain reserves of oil containing varying amounts of EPA.

Certain marine microorganisms are known to contain EPA. For example,Yazawa et al., J. Biochem, 103:5-7 (1988), found 88 strains ofgram-negative bacteria which produced EPA. U.S. Pat. No. 4,615,839 (Setoet al.) discloses the cultivation of monocellular green algae in openpools followed by recovery of EPA from those microalgae.

While omega-3-fatty acids are known to have medicinal utility, there areproblems associated with their use. Because of their association withfish oils, there is often a fishy odor and unpleasant taste associatedwith these acids. Additionally, although fish oils do contain EPA, manyof these oils cannot be consumed by humans due to the presence ofattendant contaminants, such as PCB, as well as a high concentration ofoxidation-sensitive polyunsaturated fatty acids, some of which exhibitbioactivities which are different from, and even antagonistic to, EPA.Furthermore, oils from many fish, particularly fish from tropical zonewaters, also contain significant quantities of arachidonic acid whichexhibits a biological effect antagonistic to EPA. While production ofomega-3-fatty acids in microorganisms would eliminate the contaminantproblems, no commercially acceptable and economically feasible method ofproducing large quantities of these acids in microorganisms has beenavailable.

Isotopically labelled EPA would be of great benefit in elucidating thepathway of omega-3-fatty acid metabolism. However, labelled EPA insufficient quantities to perform such research has not heretofore beenobtainable.

Accordingly, it is an object of the present invention to produce EPA inmicroorganisms by a commercially feasible method to obtain commerciallyacceptable yields.

Further, it is an object of the present invention to produceisotopically labelled EPA from this cultivation process in amountssufficient to study omega-3-fatty acid metabolism.

SUMMARY OF THE INVENTION

The present invention relates to the cultivation of microorganisms in afermentor, inducing the generation of edible oils containingomega-3-fatty acids in those microorganisms and recovering those oilsand/or fatty acids. The invention also is directed to novel oils whichcontain omega-3-fatty acids but lack the additional polyunsaturatedfatty acids associated with fish oils, to diatoms having increasedamounts of omega-3-fatty acids as compared to wild type diatoms growingin the wild, and to mutant diatoms. Typically, these oils are furthercharacterized as exhibiting biphasic melting patterns. Furthermore,isotopically labelled omega-3-fatty acids and their production aredisclosed.

The present invention provides an economical method of obtaining edibleoils having favorable organoleptic characteristics containing EPAwithout significant amounts of other polyunsaturated fatty acids.Additionally, the method permits cultivation of diatoms to greater celldensities than those typically achieved by prior art processes. Theedible oils produced by this method are free of environmentalcontaminants often found in EPA-containing oils from other sources.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic representation of the biomass accumulation inNitzschia alba during its growth and oleogenic phases.

FIG. 2 illustrates the process of labelling EPA with either ¹³ C ordeuterium.

DETAILED DESCRIPTION OF THE BEST MODE OF PRACTICING THE INVENTION

In accordance with the present invention, diatoms capable of producingEPA are cultivated in a fermentor containing a nutrient solution capableof supporting the growth of such microorganisms. In their nativeenvironment, heterotrophic diatoms are found growing epiphitically onseaweed. Accordingly, sea water is an acceptable medium for the nutrientsolution. The sea water can be either natural, filtered or an artificialmix, each of which can be diluted down to 1/4 strength or concentratedto 2×. Micronutrients can be added and may be required, especially ifthe sea water is an artificial mix. Such micronutrients are known tothose of skill in the art and are readily available from commercialsuppliers. Additionally, a growth medium specifically designed forgrowing diatoms is added. A preferred growth medium is presented inTable 1. It is to be understood that variations in this growth mediumare well within the ability of skilled workers in this art.

                  TABLE 1                                                         ______________________________________                                        GROWTH MEDIUM COMPOSITION                                                     Ingredients needed for 2 × 30 L Fermentors and 2 × 350 L          Fermentors.                                                                                    Total                                                                           30 L-          350 L-                                      Recipe             Batch          Batch                                       ______________________________________                                        19 g/L I.O. (Instant Ocean ®)                                                                570    g       6.65 Kg                                     3 g/L NaNO.sub.3   90     g       1.05 Kg                                     0.5 g/L NaH.sub.2 PO.sub.4.H.sub.2 O                                                             15     g       175  g                                      0.2 g/L Na.sub.2 SiO.sub.3.5H.sub.2 O                                                            6      g       70   g                                      6 ml/L f/2 TM (trace metals)                                                                     180    ml      2.1  L                                      60 mg/L H.sub.3 BO.sub.3                                                                         1.8    g       21   g                                      6 mg/L Na.sub.2 SeO.sub.3                                                                        180    mg      2.1  g                                      10 mg/L NaF        300    mg      3.5  g                                      40 mg/L SrO.sub.2.6H.sub.2 O                                                                     1.2    g       14   g                                      150 mg/L KBr       4.5    g       52.5 g                                      0.5 g/L KCl        15     g       175  g                                      2 ml/L B.sub.6 TM (trace metals)                                                                 60     ml      700  ml                                     After Sterilization                                                           O.1 ml/L of 0.1 mg/ml B.sub.12                                                                   3      ml      35   ml                                     O.1 ml/L of 01 mg/ml Biotin                                                                      3      ml      35   ml                                     2 ml/L of 1 mg/ml Thiamine HCl                                                                   60     ml      700  ml                                     Glucose:                                                                      (1) Start with 80 g/L                                                                            6      L       70   L                                      (40% stock solution)                                                          (2) Add another 40 g/l                                                                           31             35   L                                      1 and 2 (additional                                                           6 liters over 2 days)                                                         Silicate:                                                                     Add 60 ml/liter of 1.8    L       21   L                                      100 g/liter stock                                                             solution add additional                                                       amounts of stock                                                              solution over course                                                          of reaction until                                                             1.8 L added                                                                   ______________________________________                                    

Any diatoms capable of producing EPA can be used in the presentinvention. Moreover, it is preferred to use heterotrophic diatoms. Forthe purposes of this specification, the term "heterotrophic diatoms"means those diatoms capable of growing in the dark on a particularcarbon substrate. Different carbon substrates can be used with differentspecies and such substrates can easily be determined by persons of skillin the art. Preferred genera of diatoms include Nitzschia, Cyclotellaand Navicula. Within Nitzschia, the colorless species are especiallypreferred. In particular, Nitzschia alba is especially suitable for usein the present invention. Intended to be utilized in this invention arewild strains, mutants or recombinantly constructed microorganisms whichproduce increased amounts of EPA when cultured in accordance with thepresent invention. Suitable diatoms can be isolated from the surfaces ofseaweed where they grow epiphitically. A sample of a strain of Nitzschiaalba, an especially preferred species, has been deposited with theAmerican Type Culture Collection, Rockville, Md., and been assignedAccession No. 40775.

The present invention provides for the culturing of diatoms at a muchhigher cell density than has heretofore been obtainable. Cell densityrefers to the amount of biomass present in the fermentator. Typically,cell density accumulations in open pond cultivation of diatoms is fromabout 0.2-1 grams dry weight/liter. In contrast, by applying the methodof the present invention, and cultivating the diatoms in a fermentor,biomass densities of from 40-50 grams dry weight/liter have beenobtained. Such a high cell density contributes to the enhancedproduction of edible oils containing EPA.

Together with sea water, the growth medium hereafter will be referred toas a nutrient solution. The nutrient solution typically includesavailable nitrogen. By available nitrogen is meant nitrogen in a formsuitable for diatom use in the biosynthesis of nitrogen containingmolecules. Examples of suitable forms of such nitrogen include sodiumnitrate or potassium nitrate. Sodium nitrate is preferred. To obtain anamount of diatom biomass equal to about 50 g/per liter of solution,about 3 to about 4 grams of sodium nitrate per liter of solution shouldbe provided. The nitrate can be included in the initial mediumsterilization and need not be added thereafter.

Also added to the medium, after sterilization, is a quantity of diatomssufficient to inoculate the fermentor. These initial diatoms arereferred to herein as the "seed" diatoms. Generally seed diatoms areobtained by culturing diatoms on agar plates and transferring cells fromthe agar plates to tubes containing 2-5 ml of culture medium. After aperiod of growth, the cells in the tubes are in turn used to inoculate50 ml of medium in a 250 ml shake flask. The contents of the inoculatedshake flask are used as the seed for a 2 liter fermentor. In productionruns it is preferred to use seed culture medium in volumes of from about5 to 10% of the volume of the fermentor. For example, in a 300 literfermentor from about 15 to 30 liters of seed culture medium preferablywould be added.

As the diatoms are being cultivated they are fed both silicate andcarbon. A preferred form of silicate is metasilicate, Na₂ SiO₃.Metasilicate has both a 5 and a 9 hydrate form. Either form isacceptable for use in the present invention. In a nutrient solutionhaving a typical pH and salinity permitting the growth of diatoms,metasilicates irreversibly form a polymeric precipitate atconcentrations in excess of about 250 mg/l. Such precipitates areunacceptable as they make the silicates unavailable for use by thediatoms. This previously unsolved problem is overcome in the presentinvention where from about 5 to about 7 grams of metasilicate per literof nutrient solution desirably is added to the fermentor. Theundesirable precipitation is avoided by feeding the metasilicate intothe fermentor at a controlled rate. The metasilicate can addedincrementally in a fed batch mode. Preferably it is added in acontinuous gradient feed. A continuous gradient feed is a slower rate ofaddition than a fed batch mode, as will be understood by those of skillin the art. Those of skill in the art, in possession of this invention,can easily determine without undue experimentation suitable rates ofsilicate addition.

During the growth phase of the diatoms the ratio of silicate to carbonpreferably is kept constant. Therefore, these two nutrients can be fedto the batch together at intervals throughout the cultivation. Usingglucose as an example of a carbon source, the ratio of glucose tometasilicate desirably is from about 10 grams to 35 grams of glucose pergram of metasilicate. A particularly preferred ratio is about 20 gramsof glucose per gram of metasilicate. Those of skill in the art caneasily calculate acceptable ratios using other carbon sources, such ashydrolyzed whey, or starch.

Alternatively, the carbon source can be added batch-wise, i.e. enoughcarbon source for a complete batch is added at the beginning of thefermentation. If this alternative is chosen, the metasilicate is addedslowly to the fermentation, effectively controlling the rate of growthof the diatoms. Typical growth rates will comprise a doubling of thebiomass every 4-8 hours.

While any type of fermentor can be used with the present invention,stirred-pot fermentors with conventional Rushton turbine-agitation are apreferred embodiment. Such turbines agitate by rotating a shaft havingprotruding flat blades for maximum aeration. Preferably the speed ofrotation is kept to a speed of less than 250 cm/sec at the tip of theshaft. Maintaining the speed at less than 250 cm/sec reduces thelikelihood of shearing, or otherwise damaging, the diatoms. Anespecially preferred fermentor for large-scale cultivation is anair-lift fermentor. Such fermentors are well known to those of skill inthe art and eliminate potential shear damage.

According to the process of the present invention, heterotrophic diatomsare cultivated in fermentors as described above. While phototrophicmicroorganisms typically produce some EPA when cultivated in, forexample, open ponds, it unexpectedly has been found that heterotrophicdiatoms can be induced to enter an oleogenic phase wherein they producea single cell oil containing EPA. FIG. 1 demonstrates the increasedproduction of biomass during oleogenesis. From about 40 to 50% of thisbiomass can be attributed to oil production. Induction of oleogenesiscan be triggered by depriving the microorganism of certain nutrients. Inparticular, it is known that limiting the availability of nitrogentriggers oleogenesis in many oil producing microbes. Moreover, limitingthe availability of silicon to diatoms is known to trigger oleogenesis.Borowitzka, "Micro-Algal Biotechnology", Cambridge University Press(1988). However, in the present invention it has been discovered thatthe timing of the imposition of a silicon deficiency substantiallyincreases the production of edible oil containing EPA by the diatom.

After about 24 to 48 hours of cultivation, the diatoms have depleted theavailable nitrogen in the growth medium. At this time, they typicallyhave achieved a biomass density of 20 to 30 g/l which can be measured asthe mass of the freeze dried pellet of cells from a known volume ofculture. Of course, this time period is somewhat flexible as it dependsin part on the amount of nitrogen initially added and on the rate ofsilicon feed. For several hours after nitrogen depletion occurs,silicate and glucose continues to be fed to the diatoms. While theseadditional nutrients can be added either continuously or incrementally,it is preferable to add the nutrients incrementally. Generally, the timeperiod of this subsequent silicate feeding will be from about 12 toabout 24 hours and is terminated when about 5 g/l of metasilicate, intotal, has been added to the culture. The diatoms then enter anoleogenic phase wherein enhanced amounts of edible oils containing EPAare more rapidly synthesized. The oleogenic phase can be continued forvarying amounts of time but preferably is from about 12 hours to about36 hours duration. Preferably the oleogenic phase will be permitted tocontinue for about 24 hours. During this phase EPA is produced as asingle cell oil. For the purposes of this specification, single cell oilmeans a triglyceride product of a unicellular microorganism. Theparticular length of time of the oleogenic phase will depend upon thetype of microorganism cultivated and the available nutrient supply andcan be determined by those of skill in the art. In the case of Nitzschiaalba the yields begin to decrease if this stage is longer than about 24to 36 hours. Harvesting to obtain the oil containing EPA can occurimmediately following the oleogenic phase.

An oxygen concentration greater than that required by aerobicrespiration of the cells enhances diatom growth and EPA synthesis. Theelevated level of oxygen is provided by high aeration rates, direct O₂sparging or fermentor pressurization. There is a direct correlationbetween dissolved oxygen concentration and EPA synthesis because O₂ is asubstrate for EPA synthesis. At a dissolved oxygen concentration of 30%of air saturation, typical EPA levels in the oil are from about 2 toabout 3%. At a dissolved oxygen concentration of 50% of air saturation,the EPA content of the oil increases to about 4-5%.

The cultivation can be carried out at any temperature at which diatomscan be grown. A preferred temperature range is from about 15° C. toabout 40° C. A convenient, and economical, temperature to carry out thecultivation is 30° C. Herein lies another advantage of the presentinvention over, for example, cultivation in open ponds which aresubjected to extremes of weather. Temperatures at the lower end of theabove range tend to improve the level of EPA with respect to unsaturatedfatty acids but such temperatures also decrease the overall productivityrate. The highest productivity rates, as measured by the rate of biomassdoubling, occur at about 30° C.

The cultivation can be carried out over a broad pH range. A preferred pHis from about 7.0 to about 8.5. This pH range can be maintained by theaddition of concentrated silicate solution at a pH of about 12. If pHadjustment is required above and beyond what the addition of silicateeffects, either sodium or potassium hydroxide can be added in an amounteffective to adjust the pH.

Also encompassed by this invention are mutant strains of diatoms havingincreased amounts of EPA, lower amounts of saturated fatty acids orboth. Techniques for obtaining mutant strains, such as treating with amutagen and screening for progeny having the desired characteristics,are known to those of skill in the art. As used herein, "increased" or"lower" means an amount greater or lesser, respectively, than the amountordinarily found in wild type diatoms.

Diatoms comprising about 40% triglycerides as their biomass are aportion of this invention. Typically, wild type diatoms are found tocomprise from about 5 to about 20% triglycerides as their biomass.Because a portion of this invention lies in the recognition that diatomssuccessfully can be economically cultivated to produce large quantitiesof single cell oil, the cultivation of such diatoms to obtain any singlecell oil is contemplated to be within the scope of this invention. Forexample, wild type diatoms of the species Nitzschia alba typically haveless than about 3% EPA and 40-60% of saturated fatty acids. The samespecies in the present invention typically has from about 3-5% EPA and50% of saturated fatty acids. This increased percentage of EPA isdesirable.

Additionally, the triglycerides of the present invention exhibit abiphasic melting pattern. As diatoms are not animals such an effect isunexpected, as will be appreciated by those of skill in the art. Such amelting pattern is exhibited by dairy fats, such as butter, but has notheretofore been reported in a single cell oil from any other primaryproducer. Accordingly, single cell oils produced by the method of thepresent invention containing triglycerides exhibiting a biphasic meltingpattern also form a portion of this invention.

A preferred oil produced by the process of this invention has thefollowing fatty acid composition.

    ______________________________________                                        Fatty   14:0   16:0   18:1 18:2 18:3 20:4 20:5 Others                         Acid                                                                          % Compo-                                                                              23     33     33   2    1    1    4    3                              sition                                                                        ______________________________________                                    

As discussed above, the present invention provides a method for reliablyand consistently obtaining large quantities of an EPA-containing oil.Typically, in one embodiment of the invention the diatoms aresynthesizing at least about 20% of their biomass as edible oil. Becausethe edible oil is a single cell oil, its recovery is greatlyfacilitated. After the oleogenic phase, the diatoms can be extracted wetor dry, according to techniques known to those of skill in the art, toproduce a complex containing lipids. After extraction, this complex oflipids can be further separated to obtain EPA using known techniques.The preferred dry extraction method uses hexane as the extractingsolvent. The cells are first centrifuged, and the cell pellet frozen andlyophilized prior to extraction with hexane. Such an extraction requireslittle or no physical disruption of the cells. Extraction with thehexane at 40° C. in a volume to mass ratio of hexane to dry biomass ofabout 4:1 obtains greater than about 95% of the oil within about 0.5hour. If a wet cell paste rather than dried cells is used, then amixture of ethanol and hexane is the preferred extraction medium.

The edible oil of the present invention contains fatty acids in additionto EPA. Predominantly, these other lipids are of only three types,palmitic (16:0), oleic (18:1) and myristic (14:0), thereby simplifyingthe purification process. In contrast, fish oils contain a wide varietyof fatty acids in addition to EPA.

Quantities of EPA of sufficient purity and amount to perform research onEPA metabolism can be obtained by the method of the present invention.Accordingly, by including an isotope in the nutrient solution, labelledEPA will be synthesized by the diatoms and can be recovered. If thelabels are of the type known as stable isotopes such as deuterium orcarbon 13 or radioisotopes such as tritium or carbon 14, the EPA willincorporate those labels and can be used in tracer studies in animals orhumans or other research. It is to be understood that in addition toproviding a labelled carbon substrate such as ¹³ C-glucose or ¹⁴C-glucose to a heterotrophic diatom grown in the absence of lightsources, ¹³ CO₂, or ¹⁴ CO₂ can be provided to an autotrophicphotosynthetic diatom. In both instances D₂ O or ³ H₂ O can be supplied.Autotrophic diatoms also must be exposed to a light source of sufficientintensity to facilitate photosynthesis. Suitable photosynthetic speciesinclude those from the genus Cyclotella, Navicula, Phaeodactylum andMonodus. These organisms are preferred for the production of labelledEPA as they are autotrophic and contain higher levels of EPA thanNitzschia alba.

The present invention also includes food products, dietary supplementsand cosmetics which contain EPA produced by the methods disclosedherein. Foods or dietary supplements containing EPA are believed to beeffective in reducing coronary disease.

The oil produced by the methods of this invention, and the EPA recoveredtherefrom, also has beneficial effects, at least in part, in thetreatment of skin disorders such as psoriasis. The use of EPA from fishoil has been reported to have a beneficial effect on skin lesions causedby psoriasis. Accordingly, cosmetics containing the single cell oil ofthis invention or the EPA recovered therefrom are included within thescope of this invention. In particular, skin treatments, lotions orcreams containing the single cell oil are contemplated. Such would havean olfactory advantage over fish oils containing EPA and also would notpossess the other contaminants found in fish oil.

The present invention having been generally described, reference is hadto the following nonlimiting specific example.

EXAMPLE

Into a conventional 30 liter stirred tank fermentor (STF) is added thenutrient medium of Table 1, exclusive of the vitamins, glucose andsilicate. The fermentor is equipped with a Rushton-type turbineagitator. The STF and the medium are sterilized. After cooling themedium to about 30° C., the vitamins are added, followed by the additionof sufficient amounts of 40% glucose syrup to provide a glucoseconcentration of about 80 g/l. Concentrated sodium metasilicatepentahydrate (100 g/l) is then added to provide a total silicateconcentration of about 200 mg/l. Next, the inoculating amount of cultureis added in an amount approximately equal to 5% of the total volume ofthe fermentor, e.g. 1.5 liters/30 liters. Agitation is commenced withthe tip speed set to 85-90 cm/sec and air sparging at 1 VVM started.Over about 16 hours an additional charge of concentrated metasilicate(0.53 liters) is added and the agitation speed increased to 126 cm/sec.Over about the next 24 hours, more concentrated silicate (0.83 liters)and glucose (3.0 liters) are added. Agitation speed again is increasedto about 180-185 cm/sec. Over about the next 3 hours an additional 0.15liters of concentrated metasilicate is added. Thus, the total amount ofmetasilicate added is about 156 grams or about 1.6 liters ofconcentrated solution. At about 48 hours additional glucose (about 3liters) is added, for a total glucose addition of about 4.8 Kg or about12 liters of 40% glucose syrup. The culture is permitted to grow for anadditional 16 hours, maintaining the agitation speed and aeration rate.Then, the fermentor is harvested using a Sharples continuous flowcentrifuge producing a biomass density of approximately 45-48 grams dryweight per liter. The resulting pellet, about 20-38% solids, is removedand frozen to about -20° C. A vacuum tray drier is used to remove waterfrom the pellet. The single cell oil pellet then is extracted withhexane. The hexane subsequently is removed by distillation leaving theextracted single cell oil.

We claim:
 1. A method of producing a single cell edible oil containingeicosapentaenoic acid (EPA) comprising: cultivating heterotrophicdiatoms in a fermentor containing a nutrient solution including silicateand available nitrogen, inducing said diatoms to enter an oleogenicphase wherein said diatoms are synthesizing at least about 20% of theirbiomass as edible oil and recovering said edible oil.
 2. The method ofclaim 1, further comprising feeding silica to said microorganisms, saidfeeding being initiated prior to inducing said oleogenic phase andcontinuing after said diatoms have depleted said available nitrogen. 3.The method of claim 2 wherein said diatoms comprise Nitzschia sp.
 4. Themethod of claim 3 wherein said diatoms comprise colorless species ofNitzschia.
 5. The method of claim 1 further comprising maintaining anoxygen concentration greater than that required by aerobic respirationof the cells in said fermentor at least from the time of said oleogenicphase initiation to harvesting.
 6. The method of claim 5 wherein saidcultivation is carried out at a temperature of from about 15° C. toabout 40° C.
 7. The method of claim 6 wherein said temperature is about30° C.
 8. The method of claim 5 wherein said cultivation is at a pH offrom about 7.0 to about 8.5.
 9. The process of claim 5 wherein saidinduction comprises withholding silicate from said nutrient solution.10. The process of claim 5 wherein the length of time of said oleogenicphase is from about 12 hours to about 36 hours.
 11. The process of claim5 further comprising causing a nitrogen deficiency in said fermentorfrom about 12 to about 24 hours prior to withholding silicate from saiddiatoms.
 12. The process of claim 5 wherein said fermentor contains amedium comprising sea water.
 13. The process of claim 12 wherein saidmedium further comprises micronutrients.
 14. The process of claim 5,wherein said fermentor comprises a stirred pot fermentor having turbineinduced agitation.
 15. The process of claim 14, wherein the speed ofsaid turbine at its tip is less than about 250 cm/sec.
 16. The processof claim 5, wherein said fermentor is an air-lift fermentor.
 17. Theprocess of claim 5 wherein said dissolved oxygen comprises about 50% ofair saturation.
 18. The process of claim 5 wherein said silica comprisesmetasilicate.
 19. The process of claim 18 wherein said metasilicate isadded to said fermentor in an amount of from about 5 grams to about 7grams per liter of culture medium.
 20. The process of claim 19 whereinsaid metasilicate is added to said fermentor in a continuous gradientfeed or a fed batch mode.
 21. The process of claim 20 wherein saidmetasilicate is fed to said diatoms in conjunction with a carbon source.22. The process of claim 21 wherein said carbon source comprisesglucose.
 23. The process of claim 22 wherein the ratio of said glucoseto said metasilicate is from about 10 grams to about 35 grams of glucoseper gram of metasilicate.
 24. The process of claim 23 wherein said ratiois about 20 grams of glucose per gram of metasilicate.
 25. The processof claim 5 wherein said nutrient solution further comprises a carbonsource selected from glucose, hydrolyzed whey or hydrolyzed starch. 26.The process of claim 25 wherein said nitrogen is in the form of sodiumnitrate or potassium nitrate.
 27. The process of claim 26 wherein saidnitrogen is of the form of sodium nitrate and said sodium nitrate ispresent in said nutrient solution at a concentration of from about 3grams to about 4 grams per liter of solution.
 28. A process forproducing EPA comprising(a) cultivating colorless diatoms in a fermentorcontaining a nutrient solution having at least available nitrogen, (b)maintaining in said nutrient solution a dissolved oxygen content of fromabout 10% to about 70% of air saturation, (c) adding to said nutrientsolution glucose and metasilicate wherein the ratio of glucose tometasilicate is from about 10:1 to about 35:1 and from about 5 to about7 grams of metasilicate are added per liter of said nutrient solution,said mixture added after said diatoms have depleted the availablenitrogen in said nutrient solution, (d) maintaining the temperature ofsaid nutrient solution at from about 15° C. to about 40° C., (e)maintaining the pH of said nutrient solution at from about 7.0 to about8.5, (f) inducing an oleogenic phase in said diatoms by ceasing saidaddition of metasilicate, and (g) recovering said oil containing saidEPA after said oleogenic phase is complete.
 29. The process of claim 28wherein said diatom comprises Nitzschia alba.
 30. The process of claim29 wherein said dissolved oxygen content is at least 50% of airsaturation, said ratio of glucose to metasilicate is about 20:1 and isadded incrementally throughout about 12 to about 24 hours after saidnitrogen-depletion, said temperature is about 30° C., and said oleogenicphase occurs over approximately a 24 hour period.
 31. A method forobtaining increased yields of EPA from cultured diatoms in a fermentorcontaining a nutrient solution comprising culturing said diatoms to acell density of from about 40 to about 50 grams per liter of saidnutrient solution, inducing an oleogenic phase in said diatoms whereinsaid EPA is synthesized and recovering said EPA.
 32. The method of claim31 wherein said diatom is an autotrophic atom.
 33. The method of claim32 wherein said autotrophic diatom is Nitzschia sp. or Cyclotella sp.34. The method of claim 31 wherein said diatom is a heterotrophicdiatom.
 35. The method of claim 34 wherein said diatom comprisesNitzschia sp. or Cyclotella sp.
 36. The method of claim 35 wherein saidNitzschia is a colorless species.
 37. The method of claim 36 whereinsaid colorless Nitzschia is Nitzschia alba.